The goal of this research project is to determine the process of assembly of proteins in the Drosophila Z-band, a structural component of the myofibril, by using a combination of developmental and genetic approaches. We have isolated and purified intact Z-discs from Drosophila flight muscle and are developing a family of monoclonal antibodies to several constituent proteins. These antibodies will be used as molecular probes to examine the sequence of assembly of Z-band proteins appearing during the development of fibrillar flight muscles. Proteins localization at various stages of myofibrillogenesis will be carried out by indirect immunofluorescence microscopy and immunoelectron microscopy. Monoclonal antibodies will also be used to examine several flightless mutants of Drosophila with structurally disturbed Z- bands to assess how a mutant gene product influences the assembly of normal proteins. Immunocytochemical analysis will identify Z-band proteins missing from abnormal Z-bands. Two dimensional gel electrophoresis experiments will extend these studies to assess whether z-band proteins whcih are not assembled but are detectable in the cell by immunoblot analysis are normal or altered. Finally, in vitro translation products of RNA from mutant muscles will be examined to determine which proteins are actually being synthesized by the cell. We expect, in many cases, that an examination of translation products will indicate one aberrant or missing protein, the product of the mutant gene, whole several components may be absent or reduced in the muscle tissue. These proteins are likely to depend on the mutant protein for proper assembly. By studying several strains of flightless mutants which affect Z-band structure in different ways, we hope to identify protein associations which establish and maintain the normal Z-band lattice. It is anticipated that the studies proposed to gather information on the assembly of the Drosophila Z-band will serve as a model for understanding the Z-band in vertebrates and might have long term application to certain myopathies which have well described Z- band abnormalities.